Production of human mutated proteins in human cells by means of homologous recombination

ABSTRACT

The invention concerns a process for the production of muteins of eukaryotic polypeptides in eukaryotic cells by means of homologous recombination. The invention additionally concerns a process for the production of human cells which are suitable for the production of human mutated proteins. Finally the invention concerns the human cells produced by the process and mutated human proteins obtainable therefrom as well as pharmaceutical preparations which contain these muteins.

[0001] The invention concerns a process for the production of muteins ofeukaryotic polypeptides in eukaryotic cells by means of homologousrecombination. The invention additionally concerns a process for theproduction of human cells which are suitable for the production of humanmutated proteins. Finally the invention concerns the human cellsproduced by the process and mutated human proteins obtainable therefromas well as pharmaceutical preparations which contain these muteins.

[0002] The production of recombinant human proteins in large amounts isknown in the field of biotechnology. Proteins obtained in this mannercan be used as therapeutic agents. The recombinant production of mutatedhuman proteins which differ from corresponding natural human proteins bydeletion, addition or/and substitution of individual amino acids orwhole peptide sections is also known.

[0003] Especially for pharmaceutical applications it is often desirableto produce human polypeptides in eukaryotic cells since, in contrast topolypeptides produced in prokaryotic cells such as E. coli, these areglycosylated and therefore differ less from the polypeptides that occurendogenously in the body so that the occurrence of undesired sideeffects such as for example increased immunogenicity or poor toleranceis less frequently observed.

[0004] Mutated human proteins have been previously produced byheterologous recombinant gene expression. For this a nucleic acidconstruct is introduced into the desired eukaryotic cell which containsthe nucleic acid sequence coding for the mutated polypeptide under thecontrol of a promoter and a selection marker gene. In this process thenucleic acid construct is integrated site-unspecifically into the genomeof the cell.

[0005] In this heterologous recombinant gene expression undesired anddisadvantageous processes can frequently occur due to thesite-unspecific integration. For example mutations and especiallydeletions in the sequence coding for the protein can occur during theprocess of integration into the genome. Furthermore the integration cantake place at a site in the genome at which Cis elements are locatedwhich have a repressing effect on the expression control sequence of thenucleic acid construct and as a result of which cells are obtained witha reduced production output for the recombinant protein. An integrationof the expression construct into an important gene for the cell leadseither to death of this cell or to a recombinant cell with functionaldisorders which can, among others, result in a reduced yield of therecombinant protein.

[0006] The insertion can also lead to a reduced stability of the cellsobtained in this manner so that over a long period they lose theirability to express the recombinant protein.

[0007] The object of the present invention was therefore to provide aprocess for the production of muteins of eukaryotic polypeptides with aglycosylation which is as similar as possible to that of the naturalprotein, in a stable production cell and in good yields and thus atleast partially eliminate the disadvantages of the prior art.

[0008] This object is achieved according to the invention by a processfor the production of muteins of eukaryotic polypeptides wherein

[0009] (i) a nucleic acid molecule capable of homologous recombinationis introduced into eukaryotic cells which contain a target nucleic acidsequence coding for an endogenous target polypeptide, the said nucleicacid molecule comprising

[0010] (a) at least one sequence section which is homologous tosequences in the gene locus of the target nucleic acid sequence and,compared to the endogenous target nucleic acid sequence, has a mutationin the coding region of the mature polypeptide and

[0011] (b) a nucleic acid section coding for a selection marker,

[0012] (ii) the cells are cultured under such conditions that ahomologous recombination of the introduced nucleic acid molecule takesplace whereby the cell contains a mutated target nucleic acid sequenceafter the homologous recombination which is able to express a mutein ofthe target polypeptide,

[0013] (iii) the cells, in which a homologous recombination has takenplace, are selected and

[0014] (iv) the mutein is isolated from the cells or/and the cellsupernatant.

[0015] Mutated eukaryotic proteins and in particular mutated humanproteins can be produced in a homologous cell by the process accordingto the invention. Surprisingly this enables a mutated protein to beobtained in high yields with a very similar glycosylation pattern tothat of the natural protein. An advantage of the process according tothe invention is that a protein can be mutated in a eukaryotic cell andthis mutein is synthesized by this cell like the protein of the cellthat occurs endogenously. A further advantage of the process accordingto the invention is that the properties of the resulting cells thatproduce the mutated protein are not disadvantageously altered due to asite-unspecific gene integration. Thus the genome of the cell is notchanged in any manner apart from the gene locus of the protein to beexpressed and hence the associated adverse effects can be excluded.

[0016] The human mutated protein produced by the process according tothe invention differs from the corresponding natural protein bydeletion, addition or/and substitution of individual amino acids orwhole peptide sections. Muteins are preferably produced which havemutations at the N-terminus and/or at the C-terminus such as e.g.deletions, insertions, substitutions or/and fusions with other e.g.human proteins.

[0017] The muteins according to the invention are preferablynon-naturally occurring polypeptides and differ from allelic variationsof the polypeptide to be mutated which occur naturally in other startingcells by at least one amino acid. Non-naturally occurring muteinsparticularly preferably differ by deletions, additions or/and insertionsof individual amino acids or peptide sections from naturally occurringallelic variations.

[0018] The cell used in the process according to the invention is anarbitrary eukaryotic cell which has at least one endogenous copy of thetarget gene to be mutated. The cell is preferably a human cell,particularly preferably an immortalized human cell such as a HeLa cell,a Namalwa cell or a HT1080 cell.

[0019] It was surprisingly found that when starting cells are used whichcontain an increased number of chromosomes on which the target gene islocated, cells can be produced by homologous recombination which producean increased yield of mutated human proteins compared with cells whichonly contain two copies of the target gene. Examples of such startingcells are tumour cell lines with genetic rearrangements such as HeLaS3(Puck et al., J.Exp.Med. 103 (1996), 273-284) and Namalwa (Nadkarni etal., Cancer 23 (1969), 64-79) which contain an increased number ofcopies of the chromosome 7.

[0020] An endogenous gene activation of the mutated target gene can becarried out to further improve the expression of the mutatedpolypeptide.

[0021] For this additional sequences can be introduced into the genomewhich positively influence the expression yield in which for example theendogenous expression control sequence of the target nucleic acidsequence is replaced at least partially by a heterologous expressioncontrol sequence. This heterologous expression control sequence cancontain a heterologous promoter or/and enhancer, the heterologousexpression control sequence preferably contains a viral promoter, inparticular a CMV promoter. Replacement of the endogenous promoter notonly enables the expression to be increased but allows synthesis of themutein when a suitable promoter is used. The heterologous promoter canbe a regulatable or constructive promoter. In addition this can be usedto inactivate Cis elements that have a repressive effect on theendogenous promoter. This can also lead to an increase in yield.

[0022] The nucleic acid molecule introduced into the starting cellcomprises at least one sequence section which allows an integration byhomologous recombination in the locus of the target gene and is suitablefor introducing the mutation in the coding region of the mature targetpolypeptide. The nucleic acid molecule preferably contains two flankingsequences which are homologous to regions of the target gene locus. Theflanking sequences preferably each have a length of at least 150 bp andcontain regions from the sequences of the target gene locus coding forthe mature target polypeptide which are modified compared to the nativesequence.

[0023] In addition the nucleic acid molecule contains a selection markergene. This can be any suitable selection marker gene for eukaryoticcells which leads to a selectable phenotype on expression e.g.antibiotic resistance, auxotrophy, expression of a surface protein etc.The neomycin phosphotransferase gene is a particularly preferredselection marker gene.

[0024] In addition the nucleic acid molecule can optionally contain anegative selection marker gene e.g. a HSV thymidine kinase gene whoseexpression destroys cells in the presence of a selective agent.

[0025] If an amplification of the modified target gene in the cell isdesired, the nucleic acid molecule contains an amplification gene.Examples of suitable amplification genes are dihydrofolate reductase,adenosine deaminase, ornithine decarboxylase etc. The dihydrofolatereductase gene is a particularly preferred amplification gene.

[0026] When the amplification gene is present, the mutated targetnucleic acid sequence can be amplified after the homologousrecombination in order to increase the number of copies in the cell.

[0027] The process according to the invention enables the mutation ofall endogenous genes present in the genome of the cell used. The targetnucleic acid sequence is preferably a tissue plasminogen activator(tPA), erythropoietin, insulin, tumour necrosis factor, interleukin orinterleukin receptor sequence. The mutein obtained by the processaccording to the invention is particularly preferably a polypeptidewhose biological properties differ from those of the correspondingnatural protein, such as a polypeptide derived from t-PA comprising theK2 and P domains of t-PA (EP 0 382 174).

[0028] The known techniques can be used to isolate the mutein. Themutein is preferably isolated from the supernatant of cells cultured insuspension. Cells that can be cultured in suspension are especiallyadvantageous for a large-scale production. This considerably simplifiesthe transfers of cultured cells that are necessary during the course ofthe production process. This leads to a considerable saving ofproduction time and production resources and hence significantly reducescosts. The mutein is particularly preferably isolated from thesupernatant of cells cultured in serum-free medium. The mutein can beisolated more simply and cheaply from cells cultured in serum-freemedium in contrast to cells cultured with serum since fewer purificationsteps are necessary.

[0029] A further subject matter of the present invention is a mutatedhuman polypeptide from a human cell obtainable by one of the processesdescribed above which is distinguished by human glycosylation and theabsence of polypeptides that are foreign to the species. The absence ofpolypeptides that are foreign to the species means less than 3% byweight impurities of polypeptides foreign to the species, preferablyless than 1% by weight and most preferably less than 0.1% by weightrelative to the amount of the desired protein.

[0030] A further subject matter of the invention is a process for theproduction of a human cell which expresses a mutein of a human targetpolypeptide which is characterized in that

[0031] (i) a nucleic acid molecule is introduced into human cells whichcontain a target nucleic acid sequence coding for an endogenous targetpolypeptide, the said nucleic acid molecule comprising

[0032] (a) at least one sequence section which is homologous tosequences in the gene locus of the target nucleic acid sequence and,compared to the endogenous target nucleic acid sequence, has a mutationin the coding region of the mature target polypeptide,

[0033] (b) optionally a heterologous expression control sequence for thetarget nucleic acid sequence and

[0034] (c) a nucleic acid section coding for a selection marker,

[0035] (ii) the cells are cultured under such conditions that ahomologous recombination of the introduced nucleic acid molecule takesplace whereby the cell contains a mutated target nucleic acid sequenceafter the homologous recombination which is able to express a mutein ofthe target polypeptide,

[0036] (iii) the cells, in which a homologous recombination has takenplace, are selected and

[0037] (iv) the cells selected in this way are isolated.

[0038] In a preferred embodiment the nucleic acid molecule additionallycontains an amplification gene and the mutated target nucleic acidsequence is amplified after the homologous recombination.

[0039] A further subject matter of the invention is a human cellobtainable by a process as described above which contains at least oneendogenous gene coding for a mutated human polypeptide.

[0040] The cell according to the invention can be cultured undersuitable culture conditions and it is preferably a cell that grows insuspension and particularly preferably a cell that grows in serum-freemedium.

[0041] A further subject matter of the invention is the use of a humancell produced by a process as described above for the production of amutein of a human polypeptide.

[0042] Yet a further subject matter of the invention is a pharmaceuticalpreparation which is characterized in that it contains a mutein asdescribed above as the active substance optionally together with otheractive substances or/and common pharmaceutical carriers, auxiliarysubstances or additives.

EXAMPLE

[0043] Construction of a t-PA mutant which contains the K2 and Pdomains:

[0044] a) Vector construction

[0045] The targeting vector is composed of the following elements(listed in the 5′-3′ sequence):

[0046] A: a 6 kb BgIII fragment which contains about 3.5 kb of the 5′upstream region of the t-PA gene (Friezner et al. 1986, JBC 261 (15):6972)

[0047] B: an approximately 5.2 gene activation sequence (as an AgeIfragment) which contains the neomycin phosphotransferase (NEO) geneunder the control of the RSV promoter and the late polyadenylation siteof SV40 as the terminator, a gene coding for an arginine mutant of themurine dihydrofolate reductase (DHFR) (Simonsen et al., Proc. Natl.Acad. Sci. USA 80 (1983), 2495) under the control of the early SV40promoter and the early SV40 polyadenylation site as the terminator(Kaufmann et al., Mol. Cell. Biol. 2 (1982), 1304; Okayama et al., Mol.Cell. Biol. 3 (1983), 280 and Schimke, J. Biol. Chem. 263 (1988), 5989)and the cytomegalovirus (CMV) promoter (Boshart et al., Cell 41 (1995),p 21)

[0048] C: an approximately 200 bp fragment isolated from the t-PA cDNAwhich corresponds to the nucleotide positions 1-199 and which containsthe coding region for the signal sequence and the first three aminoacids of the mature t-PA (Pennica et al. 1983, Nature 301:214)

[0049] D: an approximately 1.5 kb EcoRI fragment which contains a largepart of the intron G of the tPA gene (Friezner et al. op.cit., Ny et al.1984, PNAS 81:5355).

[0050] These elements were isolated from the appropriate startingmaterials and assembled by means of PCR and suitable fusion PCR primers.Subsequently the fused elements were ligated into pBR322 and introducedinto E.coli. Alternatively the fragments can also be cut out from therespective starting materials and ligated via linkers.

[0051] b) Human cell line

[0052] HeLa was used as the cell line to carry out the endogenous geneactivation in which it was shown that the transcription of the t-PA genecan be induced by the addition of phorbol myristate acetate (Waller andSchleuning 1985, J. Biol. Chem. 260:6354). After introduction of thetargeting vector by means of electroporation, the cells containing thevector were selected by addition of G418. The cells which, as a resultof homologous recombination, secreted a polypeptide with the t-PAdomains K2 and P were identified by testing the supernatant of the cellswith an ELISA (Imubind-Total tPA, American Diagnostics) which is able todetect the expression of the desired polypeptide.

1. Process for the production of muteins of eukaryotic polypeptideswherein (i) a nucleic acid molecule capable of homologous recombinationis introduced into eukaryotic cells which contain a target nucleic acidsequence coding for an endogenous target polypeptide, the said nucleicacid molecule comprising (a) at least one sequence section which ishomologous to sequences in the gene locus of the target nucleic acidsequence and, compared to the endogenous target nucleic acid sequence,has a mutation in the coding region of the mature target polypeptide and(b) a nucleic acid section coding for a selection marker, (ii) the cellsare cultured under such conditions that a homologous recombination ofthe introduced nucleic acid molecule takes place whereby the cellcontains a mutated target nucleic acid sequence after the homologousrecombination which is able to express a mutein of the targetpolypeptide, (iii) the cells, in which a homologous recombination hastaken place, are selected and (iv) the mutein is isolated from the cellsor/and the cell supernatant.
 2. Process as claimed in claim 1, whereinthe cell is a human cell.
 3. Process as claimed in claim 2, wherein thecell is a HeLa cell, a Namalwa cell or a HT1080 cell.
 4. Process asclaimed in one of the claims 1 to 3, wherein a starting cell is usedwhich contains the target nucleic acid sequence on multiple chromosomes.5. Process as claimed in one of the previous claims, whereinadditionally the expression of the target nucleic acid sequence isactivated by introducing a heterologous expression control sequence. 6.Process as claimed in claim 5, wherein the heterologous expressioncontrol sequence is a viral promoter and in particular a CMV promoter.7. Process as claimed in one of the previous claims, wherein the nucleicacid section coding for the selection marker is a neomycinphosphotransferase gene.
 8. Process as claimed in one of the previousclaims, wherein the nucleic acid molecule introduced into the celladditionally contains an amplification gene and the mutated targetnucleic acid sequence is amplified after the homologous recombination.9. Process as claimed in claim 8, wherein a dihydrofolate reductase geneis used as an amplification gene.
 10. Process as claimed in one of theprevious claims, wherein the target nucleic acid sequence is a tissueplasminogen activator (t-PA), erythropoietin, insulin, tumour necrosisfactor, interleukin or interleukin receptor sequence.
 11. Process asclaimed in one of the claims 1 to 9, wherein the mutein is a polypeptidederived from t-PA comprising the K2 and P domains of t-PA.
 12. Processas claimed in one of the previous claims, wherein the mutein is isolatedfrom the supernatant of cells cultured in suspension.
 13. Process asclaimed in one of the previous claims, wherein the mutein is isolatedfrom the supernatant of cells cultured in serum-free medium.
 14. Mutatedhuman polypeptide from a human cell obtainable by a process as claimedin one of the claims 1 to 13, characterized by human glycosylation andthe absence of polypeptides that are foreign to the species.
 15. Processfor the production of a human cell which expresses a mutein of a humantarget polypeptide, wherein (i) a nucleic acid molecule is introducedinto human cells which contain a target nucleic acid sequence coding foran endogenous target polypeptide, the said nucleic acid moleculecomprising (a) at least one sequence section which is homologous tosequences in the gene locus of the target nucleic acid sequence and,compared to the endogenous target nucleic acid sequence, has a mutationin the coding region of the mature target polypeptide, (b) optionally aheterologous expression control sequence for the target nucleic acidsequence and (c) a nucleic acid section coding for a selection marker,(ii) the cells are cultured under such conditions that a homologousrecombination of the introduced nucleic acid molecule takes placewhereby the cell contains a mutated target nucleic acid sequence afterthe homologous recombination which is able to express a mutein of thetarget polypeptide, (iii) the cells, in which a homologous recombinationhas taken place, are selected and (iv) the cells selected in this wayare isolated.
 16. Process as claimed in claim 15, wherein the nucleicacid molecule additionally contains an amplification gene and, after thehomologous recombination, the mutated target nucleic acid sequence isamplified.
 17. Process as claimed in claim 16, wherein a dihydrofolatereductase gene is used as an amplification gene.
 18. Human cellobtainable by a process as claimed in one of the claims 15 to 17 whichcontains at least one endogenous gene which codes for a mutated humanpolypeptide.
 19. Use of a human cell as claimed in claim 18 for theproduction of a mutein of a human polypeptide.
 20. Pharmaceuticalpreparation, wherein it contains a mutein as claimed in claim 14 as theactive substance optionally together with other active substances or/andcommon pharmaceutical carriers, auxiliary substances or additives.